Analysis of mini-F plasmid replication by transposition mutagenesis

Derivatives of a mini-F plasmid in which Tn3 is inserted in F deoxyribonucleic acid were obtained, and the sites of insertion for 40 of the derivatives were mapped. Tn3 was found to insert at many sites within mini-F, but most insertions were within the 43.0- to 43.7-kilobase (kb), 44.2- to 44.7-kb, and 45.9- to 46.3-kb segments. Hence, these segments are unnecessary for mini-F replication. Most of the Tn3 derivatives were similar to their parent miniplasmid with respect to copy number, stability, and incompatibility. Insertions at 45.15 kb and near 46.0 kb caused a moderate disruption of copy number control, and insertion at 47.6 kb resulted in unstable maintenance. Deletion derivatives lacking deoxyribonucleic acid between 40.3 and 44.76 kb and between 45.92 and 49.4 kb were obtained. This observation suggests either that mini-F contains a third origin, in addition to those already reported to be at 42.6 and 44.4 kb, or that the reported position of the secondary origin, 44.4 kb, is incorrect and that this origin is between 44.76 and 45.92 kb.     

Utilization of the white-rot fungus Sporotrichum pulverulentum for water purification and protein production on mixed lignocellulosic wastewaters

A process has been developed that allows a direct conversion of lignocellulosic materials into fungal biomass. The thermotolerant white-rot fungus Sporotichum pulverulentium has been used in continuous laboratory fermentations as well as in a 25 m³ batch fermentation. Fungal cell mass for feeding trials was produced and the economics of the process were estimated. The investigation shows that the process works satisfactorily on the small continuous scale as well as in the large batch culture. The process also seems easy to scale up. The economic evaluations show the conversion of solid lignocellulosic materials to protein feed is not feasible by our process unless the material to be fermented has a certain negative value. A mixed wastewater, such as the white water system in paper and fiber board mills, containing both water soluble mono- and oligosaccharides and solid lignocellulosic material, can, however, be fermented in an economically feasible way due to the combined effect of protein production and water purification. Data on the nutritional value of the product are presented in an accompanying paper.     

The beta-mannanase from "Caldocellum saccharolyticum" is part of a multidomain enzyme.

The complete sequence of a beta-mannanase gene from an anaerobic extreme thermophile was determined, and it shows that the expressed protein consists of two catalytic domains and two binding domains separated by spacer regions rich in proline and threonine residues. The amino-terminal catalytic domain has beta-mannanase activity, and the carboxy-terminal domain acts as an endoglucanase. Neither domain shows homology with any other cellulase or hemicellulase sequence at the nucleic acid or protein level.     

Overproduction of an acetylxylan esterase from the extreme thermophile “Caldocellum saccharolyticum” in Escherichia coli

Summary The xynC gene coding for an acetylxylan esterase from the extreme thermophile Caldocellum saccharolyticum was overexpressed in Escherichia coli strain RR28 by cloning the gene downstream from the lacZ promoter region of pUC18 (pNZ1447) or downstream from the temperature-inducible p _r p _l promoters of pJLA602 (pNZ1600). The protein formed high molecular weight aggregates in induced cells of RR28/pNZ1600 but not in RR28/pNZ1447. The enzyme constituted up to 10% of the total cell protein and was located in the cytoplasmic fraction of RR28/pNZ1447. The acetyl esterase was most active at pH 6.0 and 70–75° C with a half-life of 64 h at 70° C and 30 h at 80° C, respectively.Offprint requests to: P. L. Bergquist     

The importance of phenol oxidase activity in lignin degradation by the white-rot fungus Sporotrichum pulverulentum

The lignin degradation abilities of wildtype, a phenol oxidase-less mutant and a phenol oxidase-positive revertant of Sporotrichum pulverulentum were compared to determine if phenol oxidase activity is necessary for lignin degradation by white-rot fungi. The phenol oxidase-less mutant was unable to degrade kraft lignin or wood. The phenol oxidase-positive revertant, however, regained the ability of the wildtype to degrade kraft lignin and all of the major components of wood. It was found that kraft lignin and lignin-related phenols decreased cellulase and xylanase production by the phenol oxidase-less mutant. Addition of highly purified laccase increased the production of endo-1,4--glucanase in the phenol oxidase-less mutant in the presence of vanillic acid and kraft lignin. After addition of laccase to kraft lignin agar plates, the phenol oxidase-less mutant could degrade kraft lignin.It is proposed that phenol oxidase function in regulating the production of both lignin-and polysaccharide-degrading enzymes by oxidation of lignin and lignin-related phenols when S. pulverulentum is growing on wood.Abbreviation WT wildtype Sporotrichum pulverulentum Research supported by a grant from Stiftelsen Nils and Dorthi Troëdssons forskningsfond     

Abnormal Excision and Transfer of Chromosomal Segments by a Strain of Escherichia coli K-12

PB15 is an Hfr strain of Escherichia coli K-12. It arose from an F' strain carrying a temperature-sensitive F-gal by an event which blocked the detachment of F-gal in the normally reversible integration process. In PB15, the detachment of F-gal by a second mechanism can now be detected: this mechanism results in the excision and transfer of extended chromosomal segments which include the integrated F-gal; the excised segments are inferred to have circularized. Their excision, which is independent of the recA(+) allele, occurs at an unusually high rate during conjugation; a mutant F-initiator protein is suggested as the cause of this phenomenon. After their establishment in recipients, the enlarged F-genotes undergo further deletions of included donor genes by a process which is again recA(+)-independent. In Rec(+), but not in Rec(-), cells, a high proportion of the deleted fragments are rescued by integration into the recipient's chromosome.     

Analysis of a region in plasmid R386 containing two functional replicons

A miniplasmid has been obtained from R386 by ligating EcoRI fragments with a fragment carrying a kanamycin-resistance gene. It contains a 6.8-kb Eco fragment of R386 which hybridizes strongly with several IncFI plasmid DNAs but not with the primary or secondary replicons of the F plasmid. This mini-R386 is incompatible with certain IncFI plasmids, and it appears to be one example of a previously unidentified replicon widely distributed in the IncFI group. A region of R386 not closely linked to the 6.8-kb fragment is involved in copy number control of the mini-R386, and a sequence in the same region interacts with mini-F partition functions to cause incompatibility. The 6.8-kb fragment also restricts growth of T7 bacteriophage, and an adjacent fragment restricts phage T4 growth. A further R386 sequence, sharing homology with the F secondary replicon, is capable of autonomous replication. Hence R386, like F, contains at least two functional replicons.